Claims:
1) A long-acting depot injectable formulation comprising;
a) Atovaquone or Proguanil/its salt in combination with Atovaquone;
b) an oil and/or a bio-degradable polymer; and
c) a solvent,
having a therapeutic effect for at least 4 weeks, preferably 8 weeks and more preferably 12 weeks.

2) The depot injectable formulation as claimed in claim 1, wherein the atovaquone present in a concentration range of 2- 20%(wt/vol).

3) The depot injectable formulation as claimed in claim 1, wherein the Proguanil or its salt present in a concentration range of 3-15% (wt/vol).

4) The depot injectable formulation as claimed in claim 1, wherein the Atovaquone and Proguanil present in a molar ratio of 1:1 to 1: 1.5.

5) The long- acting depot injectable formulation as claimed in claim 1, wherein the formulation further comprises anti busting agent and optionally, hydrophilic polymer, solubilizing agent and/or surfactant.

6) The depot injectable formulation as claimed in claim 1, wherein the oil is selected from a group consisting of castor oil, corn oil, peanut oil, sesame oil, olive oil, palm oil, sunflower oil, soybean oil, cottonseed oil, rapeseed oil and mixtures thereof present in a concentration of 10-70% (vol/vol).

7) The depot injectable formulation as claimed in claim 1, wherein the bio-degradable polymer is selected from a group consisting of polylactides (PLA); poly(lactide-co-glycolide) (PLGA); polyanhydrides; polyorthoesters; poly(N-(2- hydroxypropyl) methacrylamide); poly(dl-lactide) (DLPLA); poly(1-lactide) (LPLA); poly(d-lactide) (DPLA); polyglycolide (PGA); poly(dioxanone) (PD0); poly(glycolide-co-trimethylene carbonate) (PGA-TMC); poly(1-lactide-co-glycolide) (PGA-LPLA); poly(dl-lactide-co-glycolide) (PGA-DLPLA); poly(1-lactide-co-dl-lactide) (LPLA-DLPLA); poly(glycolide-co-trimethylene carbonate-co-dioxanone) (PDO-PGA-TMC), poly(lactic acid-co-caprolactone) (PLACL) and mixtures thereof present in a concentration range of 10-50% (wt/vol).

8) The depot injectable formulation as claimed in claim 1, wherein the solvent is selected from a group consisting of N-Methyl-2-pyrrolidone (NMP), triacetin, Dimethyl sulphoxide (DMSO), benzyl alchol and ethylacetate present in a concentration range of 10-70% (vol/vol).

9) The depot injectable formulation as claimed in claim 5, wherein the anti-busting agent is selected from a group consisting of Polyethylene glycol-400, PLURONIC® L101, PLURONIC® L121, PLURONIC® P81, PLURONIC® P84 and PLURONIC® P85, Tween 20 and Tween 80, Span 80, Chremophore RH 40 and Chremophore RH 60 present in a concentration range of 1-10% (wt/vol).

10) The depot injectable formulation as claimed in claim 5, wherein the hydrophilic polymer is selected from a group consisting of Poly (vinyl pyrrolidone), Poly (vinyl alcohol), Poly (vinyl acetate), Poly (vinyl caprolactum), Poly (ethylene glycol) present in a concentration range of 0.01-2% (wt/vol).

11) The depot injectable formulation as claimed in claim 5, wherein the solubilizing agent is selected from a group consisting of glycerol, glycerin, dimethylacetamide, N-methyl-2-pyrrolidone, propylene glycol, polyethylene glycol 300, polyethylene glycol 400, ricinoleate based solubiliser, cyclodextrin, Kolliphor HS 15, Kolliphor EL, Kolliphor RH 60 and Polysorbate 80 present in a concentration range of 1-6% (wt/vol).

12) The depot injectable formulation as claimed in claim 5, wherein the surfactant is selected from a group consisting of poloxamer, Cremophor, Gelucire and sodium lauryl sulphate present in a concentration range of 0.02-4% (wt/vol).

13) The depot injectable formulation as claimed in claim any one of the preceding claims 1 to 12, wherein the composition is administered once in 2 to 12 weeks.

14) A method for preparing the long- acting depot injectable formulation as claimed in claim 1 comprises;
(c) Dissolving Atovaquone or Proguanil/its salt in combination with Atovaquone in a solvent to obtain a solution; and
(d) mixing an oil and/or a bio-degradable polymer, optionally mixing anti busting agent, hydrophilic polymer, solubilizing agent and/or surfactant with the solution of step a) to obtain long-acting depot injectable formulation.
, Description:FIELD OF THE INVENTION:
The present invention relates to a novel pharmaceutical composition of Atovaquone or Proguanil in combination with Atovaquone. More particularly, the invention relates to long- acting injectable composition/formulation which provides sustained depot effect of Atovaquone & Proguanil and offering prolonged therapeutic and prophylactic benefit.

BACKGROUND OF THE INVENTION:
Class II BCS drugs have high permeability but poor aqueous solubility and thus exhibit poor dissolution profile limiting its bioavailability following oral administration. In order to achieve sufficient therapeutic effect systemically, high oral dose is required to be administered to evade this limitation and to achieve desired therapeutic concentration in systemic circulation.

Atovaquone, a hydroxy-1,4-naphthoquinone, is a well-known anti-malarial and anti-pneumocystic drug, which is commercially available in different dosage forms as standalone or in combination with Proguanil since 1992, but the bioavailability of tablet formulations barely crosses 12% (23% under fat fed conditions) and for oral nano-sized suspension, bioavailability is about 23% (46-48% under fat fed condition). Fatty meal helps to emulsify the drug in the lipids present in the food. Owing to its poor hydro solubility, Atovaquone dissolution is hampered in aqueous environment of the digestive tract thus limiting its bioavailability and adding to inter-patient variance. In order to achieve therapeutic levels of Atovaquone, patient is advised to eat high fatty food, and very often this turns out be non-feasible because of severity or nature of the underlying disease.

Atovaquone (Formula I), chemical name being trans-2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone, is a hydroxy-1,4-naphthoquinone, an analog of Ubiquinone, with antipneumocystic and anti-malarial activity. It has previously been disclosed, for example, in European Patent No. 1,23,238 and 0675711 that Atovaquone is active (in animals and in vitro) against Pneumocystis (carinii) jirovecii, Plasmodia, tachyzoite and cyst forms of Toxoplasma gondii, and Eimeria spp., a causative agent for Coccidiosis. Further uses of Atovaquone for Cryptosporidiosis and Babesiosis are disclosed in European patent application no. 0496729 and U.S. Pat. No. 5,559,156 respectively.

Formula-I
For treatment as well as prophylaxis of malaria, Proguanil is co-administered with Atovaquone. Chemical structure of Proguanil is given in Formula II.

Formula-II
As mentioned in The Comprehensive Pharmacology Reference, 2007, Proguanil is having a half-life (plasma) of 12-20 hrs (oral) and approximately 100% bioavailability (oral).

Malaria is an infectious disease that causes severe morbidity and mortality with an estimated 300-500 million cases worldwide and more than 1 million deaths annually in sub-Saharan Africa alone and affected patients are of any age group. The disease is caused by a protozoan parasite of the genus Plasmodium, transmitted by mosquitoes. The most serious forms of malaria are caused by Plasmodium falciparum and Plasmodium vivax, but other species (e.g., Plasmodium ovale, Plasmodium malariae, and Plasmodium Knowlesi) can also infect humans. Control of malaria has been hampered by the spread of drug resistance in both the Plasmodium parasites and the Anopheles insect vector, and by the lack of an efficacious vaccine (Moorthy, V. S. et al., 2004. Lancet 363:150-156).

Atovaquone has been approved as standalone monotherapy for treating Pneumocystis (carinii) jirovecii infections in AIDS patients and in combination with proguanil for prophylaxis and treatment of malaria.

Atovaquone was initially granted orphan drug status in 1990 for treatment and in 1991 for prevention of Pneumocystis carinii infections in AIDS patients. Atovaquone 250mg tablet formulation (MepronTM) was approved in 1992 for treatment and prophylaxis of Pneumocystis carinii infections in AIDS patients. In 1993 Atovaquone was conferred orphan drug status for Toxoplasmosis as well but was subsequently withdrawn due to lack of clinical efficacy which was partially attributed to low bioavailability and high inter-patient variability. A nanoparticle of MepronTM suspension with improved bioavailability was launched in 1995 and the tablet formulation was then withdrawn from the market.

Numerous attempts have been made since then for increasing the bioavailability of Atovaquone by primarily focusing on particle size reduction, however, significant dissolution profile and bioavailability improvement over nano suspension is far from reach.

Various other publications such as US6018080A/US6649659B1& US20100028425 reported improving bioavailability of Atovaquone. Our previously filed application WO2014045307 reported Atovaquone-Proguanil complex with improved Atovaquone dissolution profile and its consequent bioavailability.

US20100099776A1 publication reported an oily suspension of Atovaquone comprising Atovaquone nano particles for improving bio-availability.

However, latest publications focused on preparation of long-acting depot formulations of Atovaquone to treat/prevent malaria in travelers to malaria-endemic countries.

A publication, WO2017222996A1, reported compositions of alkene esters of Atovaquone of Formula-III to treat/prevent malaria in travelers to malaria-endemic countries. The compositions are having a depot effect of about 27 days (table - 25/26).

Formula-III

Another publication, WO2018204563A1, reported intramuscularly administering an effective amount of injectable formulation of microparticles of Atovaquone once in 42 days for treating/preventing malaria.

Yet, another publication, Nature Communications (2018) 9, p. 315, reported long-acting intramuscular aqueous injectable atovaquone nanopartilcles. The formulation is effective upto 4 weeks after administration of the injection.

Existing art reported long acting depot formulations of Atovaquone alone. As reported in Antimicrobial Agents and Chemotherapy, June 1999, p. 1334–1339, Proguanil has synergistic effect with Atovaquone in treating malaria. Proguanil enhanced the ability of Atovaquone by approximately seven folds and also lowered the in vivo 50% inhibitory concentration of Atovaquone to treat/prevent malaria.

Although depot formulations of Atovaquone exist, Proguanil is to be administered frequently to potentiate Atovaquone. Moreover, since Proguanil is having lesser half-life [half-life (plasma) of 12-20 hrs (oral)]; daily doses are given throughout the season of highest malaria transmission (3-4 months) to prevent malaria.

Therefore, a single injectable dose would be preferred to ensure continuous coverage throughout the entire season and to simplify patient compliance to maximize chemo prevention, effective within hours of dosing, that eliminates noncompliance and the necessity for carrying (and taking) medication.

Therefore, the challenge remains in developing long-acting depot injectable formulation of Atovaquone or Proguanil in combination with Atovaquone to avoid frequent administration. So that once the single dose of depot formulation is administered via intramuscular/subcutaneous, it will be effective for prolonged period (i.e., for several months) for treating/preventing malaria.

Accordingly, the objective of present invention is to provide long-acting depot formulation of Proguanil in combination with Atovaquone which provides sustained depot effect to treat/prophylaxis/seasonal chemo prevention of malaria up to 12 weeks after administration.

SUMMARY OF INVENTION:
In line with the above objective, the present inventors have developed a novel a long-acting depot injectable formulation of Atovaquone or Atovaquone and Proguanil mixture (Atoguanil) having a depot effect.

Accordingly, in one aspect, the present invention provides a novel long-acting depot injectable formulation which comprises;
a) Atovaquone or Proguanil or its salt in combination with Atovaquone;
b) an oil and/or a bio-degradable polymer; and
c) a solvent,
having a therapeutic effect for at least 4 weeks, preferably 8 weeks and more preferably 12 weeks.

In another aspect, the long- acting depot injectable formulation of the present invention further comprises anti busting agent and optionally, hydrophilic polymer, solubilizing agent and/or surfactant.

In a further aspect, the present invention provides a method for preparing the long-acting depot injectable formulation comprising;

(a) Dissolving Atovaquone or Proguanil or its salt in combination with Atovaquone in a solvent to obtain a solution; and
(b) mixing an oil and/or a bio-degradable polymer, optionally mixing anti busting agent, hydrophilic polymer, solubilizing agent and/or surfactant with the solution of step a) to obtain long- acting depot injectable formulation.

DESCRIPTION OF INVENTION:
Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. To describe the invention, certain terms are defined herein specifically as follows.

Unless stated to the contrary, any of the words “including,” “includes,” “comprising,” and “comprises” mean “including without limitation” and shall not be construed to limit any general statement that it follows to the specific or similar items or matters immediately following it. Embodiments of the invention are not mutually exclusive, but may be implemented in various combinations. The described embodiments of the invention and the disclosed examples are given for the purpose of illustration rather than limitation of the invention as set forth the appended claims.

The term "subject" or "patient" encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non -human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.

The present invention is based on the pharmaceutical development of a novel long-acting depot injectable formulation of Atovaquone or Proguanil orits salt in combination with Atovaquone. The injectable formulation provides desired and sustained therapeutic effect by releasing the major content of the active ingredient of Atovaquone or atovaquone and proguanil from the formulation for at least 4 weeks, preferably 8 w eeks and more preferably 12 weeks.

Accordingly, in one of the embodiments, the present invention provides a novel a long- acting depot injectable formulation comprising;
a) Atovaquone or Proguanil or its salt in combination with Atovaquone;
b) an oil and/or a bio-degradable polymer; and
c) a solvent,
having a therapeutic effect for at least 4 weeks, preferably 8 weeks and more preferably 12 weeks.

In present invention Proguanil as such or its salt such as Proguanil hydrochloride may be used. However, the preferred one is Proguanil free base.

The ‘oil’ as referred herein may include, but not limited to castor oil, corn oil, peanut oil, sesame oil, olive oil, palm oil, sunflower oil, soybean oil, cottonseed oil, rapeseed oil and mixtures thereof. However, the preferred oil is castor oil or sesame oil.

The ‘bio-degradable polymer’ may include polylactides (PLA); poly(lactide-co-glycolide) (PLGA); polyanhydrides; polyorthoesters; poly(N-(2- hydroxypropyl) methacrylamide); poly(dl-lactide) (DLPLA); poly(1-lactide) (LPLA); poly(d-lactide) (DPLA); polyglycolide (PGA); poly(dioxanone) (PD0); poly(glycolide-co-trimethylene carbonate) (PGA-TMC); poly(1-lactide-co-glycolide) (PGA-LPLA); poly(dl-lactide-co-glycolide) (PGA-DLPLA); poly(1-lactide-co-dl-lactide) (LPLA-DLPLA); poly(glycolide-co-trimethylene carbonate-co-dioxanone) (PDO-PGA-TMC), poly(lactic acid-co-caprolactone) (PLACL) and mixtures thereof. However, the preferred one is poly(lactide-co-glycolide) also known as poly (lactic-co-glycolic acid).

Suitable solvents include N-Methyl-2-pyrrolidone (NMP), triacetin, Dimethyl sulphoxide (DMSO), benzyl alchol and ethylacetate. However, the preferred solvent is NMP.

In another embodiment, the present invention provides a novel a long-acting depot injectable formulation which comprises;
a) Atovaquone or Proguanil or its salt in combination with Atovaquone;
b) an oil and/or a bio-degradable polymer;
c) a solvent; and
d) anti-busting agent and optionally, hydrophilic polymer(s), solubilizing agent(s) and/or surfactant(s), having a therapeutic effect for at least 4 weeks, preferably 8 weeks and more preferably 12 weeks.

Anti-busting agent(s) include, Polyethylene glycol-400; Pluronics such as PLURONIC® L101, PLURONIC® L121, PLURONIC® P81, PLURONIC® P84 and PLURONIC® P85; Tweens such as tween 20 and tween 80; Spans such as Span 80; and Chremophores such as Chremophore RH 40 and Chremophore RH 60. However, the preferred anti busting agent is Polyethylene glycol-400 and PLURONIC® P85. Pluronic® P85 (Pluronic is a trademark of BASF Corp.), also referred to as Poloxamer 235, is a basic hydroxyl-terminated, difunctional block copolymer surfactant. It is a non-ionic, relatively non-toxic surfactant. Pluronic® P85 has an average molecular weight of 4600, a viscosity of 310 cps at 60 ° C, a cloud point (at a water content of 10%) of 83-89°C and a hydrophilic-lipophilic balance of 12-18.

Hydrophilic polymer(s) includes Poly (vinyl pyrrolidone), Poly (vinyl alcohol), Poly (vinyl acetate), Poly (vinyl caprolactum), Poly (ethylene glycol). However, the preferred Hydrophilic polymer is Poly (vinyl pyrrolidone), i.e., PVP-K30.

The ‘solubilizing agent’ includes but not limited to glycerol, glycerin, dimethylacetamide, N-methyl-2-pyrrolidone, propylene glycol, polyethylene glycol 300, polyethylene glycol 400, ricinoleate based solubiliser, cyclodextrin, Kolliphor HS 15 (Solutol HS 15), Kolliphor EL (Cremophor EL), Kolliphor RH 60 (Cremophor RH 60), and polysorbate 80 (Tween 80). However, the preferred solubilizing agent is Kolliphor HS 15.

The ‘surfactant agents’ include 2-(2-propoxypropoxy)ethanol (poloxamer 188), Cremophor/Kolliphor, Gelucire and sodium lauryl sulphate. However, the preferred surfactant is poloxamer 188.

In one of the preferred embodiments, the ratio of Atovaquone and Proguanil is used in 1:1 molar ratio. However, Proguanil ratio may be increased up to 1.5: 1 (proguanil: Atovaquone) as its half-life period is less and it increases the bio-availability of Atovaquone.
The concentration of the Atovaquone in the injectable formulation may be in the range of 2- 20% (wt/vol). However, the preferred range is 5-10% (wt/vol).

The concentration of the Proguanil or its salt in the injectable formulation may be in the range of 3-15% (wt/vol). However, the preferred range is 6- 8% (wt/vol).

The concentration of oil in the injectable formulation may be in the range of 10-70% (vol/vol). However, the preferred concentration is in the range of 10-50% (vol/vol).

The concentration of bio-degradable polymer in the injectable formulation may be in the range of 10-50% (wt/vol). However, the preferred concentration is in the range of 15-30% (wt/vol).

The concentration of hydrophilic polymer in the injectable formulation may be in the range of 0.01-2% (wt/vol). However, the preferred concentration is between 0.05-0.5% (wt/vol).

The concentration of solubulizing agent in the injectable formulation may be in the range of 1-6% (wt/vol). However, the preferred concentration is in the range of 2-4% (wt/vol).

The concentration of surfactant in the injectable formulation may be in the range of 0.02-4% (wt/vol). However, the preferred concentration is in the range of 0.01-1 % (wt/vol).

The concentration of the anti-busting agent in the injectable formulation may be in the range of 1-10% (wt/vol). However, the preferred concentration is in the range of 3- 6 % (wt/vol).

The concentration of solvent in the injectable formulation may be in the range of 10-70% (vol/vol). However, the preferred concentration is in the range of 20-60% (vol/vol).

In another embodiment, present invention provides a method for preparing the injectable formulation comprising the steps of;

a) dissolving Atovaquone or Proguanil or its salt in combination with Atovaquone in a solvent to obtain a solution; and
b) mixing an oil and/or a bio-degradable polymer, optionally mixing anti busting agent, hydrophilic polymer, solubilizing agent and/or surfactant with the obtained solution to obtain long- acting depot injectable formulation.

Accordingly, the Atovaquone or Atovaquone and proguanil complex is dissolved in a suitable solvent to obtain clear solution. To the obtained solution, bio-degradable polymer is added and mixed well to obtain the long- acting depot injectable formulation.

Alternatively, an oil is added in place of bio-degradable polymer to obtain the long-acting depot injectable formulation. In another alternative, oil is added along with bio-degradable polymer to obtain the long -acting depot injectable formulation.

To the injectable formulation, optionally, other pharmaceutically acceptable excipients such as anti-busting agents, hydrophilic polymers, solubilizing agent and/or surfactants may be added.

Accordingly, the Proguanil or its salt in combination with Atovaquone solution can be obtained by dissolving both Atovaquone and Proguanil or its salt in a suitable solvent and mixing the same to obtain the combined solution of Atovaquone and Proguanil or its salt.
In another alternative embodiment, Atovaquone-proguanil complex may be prepared by mixing atovaquone solution (dissolved in methylenedichloride) with proguanil solution (dissolved in methylenedichloride) to obtain Atovaquone-proguanil mixture. The mixture is concentrated, filtered and washed the cake to obtain Atovaquone-proguanil complex (Atoguanil).

Alternatively, spray dried Atoguanil (preformulated powder or granules) comprising hydrophilic polymer, solubilizing agent and surfactant may be used in place of Atovaquone-proguanil complex for obtaining the long-acting depot injectable formulation.

The obtained injectable formulation prepared as per the invention may be in clear solution or it may be in micro emulsion form.

Since the formulation is sensitive to moisture, optionally, two separate solutions may be prepared. And, these two solutions are mixed prior to injecting into patient. Alternatively, these two solutions are mixed in a syringe prior to injecting. In an additional embodiment, one solution may be prepared using Atovaquone and proguanil, hydrophilic polymer, solubilizing agent and surfactant in solvent and, second solution may be prepared using Bio-degradable polymer and/or oil in solvent and mixing the first and second solution prior to injecting into a patient.

The formulation obtained as per the invention is efficient, well-tolerated, sustained or delayed release of the active ingredients that are therapeutically effective for a number of weeks, such as at least about one week, at least about two weeks, at least about four weeks, at least about 8 weeks and at least 12 weeks. The Atovaquone and proguanil released from the depot formulation at a rate providing plasma concentration in the range of 0 to 20 µg/mL of Atovaquone and 0 to 150 ng/mL of Proguanil over 14 weeks after administration.

In yet another embodiment, the invention provides long acting depot injectable composition comprising Atovaquone or Proguanil or its salt in combination with Atovaquone, provided according to the invention for use in the treatment of seasonal chemo protection or prophylaxis of a protozoal parasitic infection or an infection caused by Pneumocystis (carinii) jirovecii in mammals, wherein the protozoal parasitic infection is selected from the group consisting of malaria, babesia, cryptosporidiosis, coccidiosis or toxoplasmosis.

The following examples, which include preferred embodiments, is intended to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention. In other embodiments, formulations for parenteral, subcutaneous, intravenous, intra-arterial, intra-thecal and Intra-peritoneal administration are contemplated, and are in no way limiting.

Examples:
Example 1:
Atovaquone and Proguanil free base complex (Atoguanil) preparation:
Charged atovaquone (50 gms) and MDC (2000 ml) in a flask and stirred for 5 min at 30 ± 2°C. Methanol (500 ml) was added and a clear transparent yellow colored solution was obtained. In separate conical flask, a solution of proguanil free base (35 gms) was prepared by dissolving in 400 ml MDC and a clear transparent solution was obtained. This solution was added to the atovaquone solution with stirring over a period of 45- 50 minutes to get the dark red color solution. Reaction was maintained at 40± 2°C for two hrs. Maximum solvent was distilled out at 40 ± 2°C. The reaction mixture was stripped with n-heptane and red colored solid was scraped from the sides of the reaction flask and 100 ml n-heptane was further added and stirred. The n-heptane was distilled out under vacuum at 40 ± 2°C for 30-40 min. One more stripping was done with 100 ml n-heptane to remove MDC and methanol completely. The red colored solid was scraped from the sides of the reaction flask and 250 ml n-heptane was added and stirred, at 30 ± 2°C for 30 minutes. Filtered the mass and washed the cake with n-heptane.
Wet cake: 85 gms. Dried the cake under vacuum oven at 60 ± 2°C for 20 hrs to obtain 80 gms dry powder.

Example 2:
Atoguanil Formulation Composition -1
Taken Atoguanil 16.6 gms (which contains 9.76 gms atovaquone and 6.83 gms proguanil) and dissolved in 30 ml of N-Methyl-2-pyrrolidone. In another flask, taken PLGA (75:25:: lactide:glycolide) 20 gms and dissolved in N-Methyl-2-pyrrolidone(25 ml). Mixed the Atoguanil solution with the PLGA solution and adjusted the quantity sufficient to make 100 ml with NMP and stirred well for an hour to obtain clear solution.

Atoguanil Formulation Composition : -1
Ingredients Quantity
Atoguanil Atovaquone 9.76 gm
Proguanil 6.83 gm
PLGA 20 gm
NMP Q.S to 100 ml

Atoguanil Formulation Composition -2
Taken Atoguanil 16.6 gms (which contains 9.76 gms atovaquone and 6.83 gms proguanil) and dissolved in 30 ml of N-Methyl-2-pyrrolidone. In another flask, taken PLGA (75:25:: lactide:glycolide) 20 gms, added 5 ml of PEG400 and dissolved in N-Methyl-2-pyrrolidone(20 ml). Mixed the Atoguanil solution with the PLGA solution and adjusted the quantity sufficient to make 100 ml with NMP and stirred well for an hour to obtain clear solution.

Atoguanil Formulation Composition : -2
Ingredients Quantity
Atoguanil Atovaquone 9.76 gm
Proguanil 6.83 gm
PLGA 20 gm
PEG 400 5 ml
NMP Q.S to 100 ml

Atoguanil Formulation Composition -3
Taken Atoguanil 16.6 gms (which contains 9.76 gms atovaquone and 6.83 gms proguanil) and dissolved in 20 ml of N-Methyl-2-pyrrolidone. In another flask, taken PLGA (75:25:: lactide:glycolide) 20 gms and dissolved in N-Methyl-2-pyrrolidone( 20 ml), added 5 ml of PEG400 and mixed well. Added the Atoguanil containing solution to the PLGA/PEG400 solution, mixed well. Added 1 ml of sesame oil, adjusted the quantity sufficient to make 100 ml with NMP and stirred well for an hour to obtain clear solution.

Atoguanil Formulation Composition : -3
Ingredients Quantity
Atoguanil Atovaquone 9.76 gm
Proguanil 6.83 gm
PLGA 20 gm
PEG 400 5 ml
Sesame Oil 10 ml
NMP Q.S to 100 ml

Example 3:
Spray Dried Atoguanil Preparation:

Added MDC (500 ml) and Kolliphor HS15 (16 g) in a flask and stirred to get clear solution. Into the obtained solution, added PVP K (30 0.6 g) and stirred for 5 min. Further charged Poloxamer 188 (1.2 g) into the solution and stirred for 5 min. In to the obtained solution added, Proguanil (27.84 g), Atovaquone (40 g) and MDC (300 ml) and stirred the reaction mixture for two hours at 25-30°C to obtain clear solution. Then the reaction mass was spray dried to get Atoguanil powder.

Spray Dried Atoguanil Formulation Composition -4
Taken Atoguanil powder 25 gms (which contains 11.67 gms atovaquone and 8.125 gms proguanil) and dissolved in 50 ml of N-Methyl-2-pyrrolidone. In another flask, taken PLGA (75:25:: lactide:glycolide) 20 gms and dissolved in N-Methyl-2-pyrrolidone(20 ml). Mixed the Spray Dried Atoguanil solution with the PLGA solution. Adjusted the quantity sufficient to make up the volume upto 100 ml with NMP and stirred well for an hour to obtain clear solution.

Atoguanil Spray Dried Formulation Composition : -4
Ingredients Quantity
Atoguanil Spray Dried Atovaquone 11.67 gm
Proguanil 8.125 gm
PLGA 20 gm
Kolliphor HS 15 3.117 gm
PVP-K30 0.117 gm
Poloxamer 188 0.233 gm
NMP Q.S to 100 ml

Spray Dried Atoguanil Formulation Composition -5
Taken Spray Dried Atoguanil powder 25 gms (which contains 11.67 gms atovaquone and 8.125 gms proguanil) and dissolved in 30 ml of N-Methyl-2-pyrrolidone. In another flask, taken PLGA (75:25:: lactide:glycolide) 20 gms and dissolved in N-Methyl-2-pyrrolidone(20 ml), added 5 ml of PEG400 and mixed well. Added the Spray Dried Atoguanil solution into the PLGA/PEG400 solution, adjusted the quantity sufficient to make up the volume upto 100 ml with NMP and stirred well for an hour to obtain clear solution.
Atoguanil Spray Dried Formulation Composition : -5
Ingredients Quantity
Atoguanil Spray Dried Atovaquone 11.67 gm
Proguanil 8.125 gm
PLGA 20 gm
PEG 400 5 ml
Kolliphor HS 15 3.117 gm
PVP-K30 0.117 gm
poloxamer 188 0.233 gm
NMP Q.S to 100 ml

Spray Dried Atoguanil Formulation Composition -6
Taken Spray Dried Atoguanil powder 25 gms (which contains 11.67 gms atovaquone and 8.125 gms proguanil) and dissolved in 20 ml of N-Methyl-2-pyrrolidone. In another taken, PLGA (75:25:: lactide:glycolide) 20 gms and dissolved in N-Methyl-2-pyrrolidone(20 ml), added 5 ml of PEG400 and mixed well. Added the Spray Dried Atoguanil solution into the PLGA/PEG400 solution, adjusted the quantity sufficient to make up the volume upto 100 ml with NMP and stirred well for an hour to obtain clear solution.

Atoguanil Spray Dried Formulation Composition : -6
Ingredients Quantity
Atoguanil Spray Dried Atovaquone 11.67 gm
Proguanil 8.125 gm
PLGA 20 gm
PEG 400 5 ml
Sesame Oil 10 ml
Kolliphor HS 15 3.117 gm
PVP-K30 0.117 gm
poloxamer 188 0.233 gm
NMP Q.S to 100 ml

Spray Dried Atoguanil Formulation Composition -7
Taken Spray Dried Atoguanil powder 16.6 gms (which contains 7.78 gms Atovaquone and 5.41 gms Proguanil) and dissolved in 20 ml of N-Methyl-2-pyrrolidone. In another taken 50 ml castor oil and dissolved in N-Methyl-2-pyrrolidone(10 ml). Added the Spray Dried Atoguanil solution into the castor oil solution, adjusted the quantity sufficient to make up the volume upto 100 ml with NMP and stirred well for an hour to obtain clear solution.

Atoguanil Spray Dried Formulation Composition
Ingredients Quantity
Atoguanil Atovaquone 7.78 gm
Proguanil 5.41 gm
Castor Oil 50 ml
Kolliphor HS 15 3.11
PVP-K30 0.116
poloxamer 188 0.233
NMP Q.S to 100 ml

Example 4:
Clinical study:
Estimation of concentration of Atovaquone and Proguanil in rat plasma samples:
Sprague–Dawley (SD) rats (weighing 220–250 g) were used for this study. Atoguanil spray dried formulation composition prepared as per example 7 was administered to the two treatment groups of S D rats as intra-muscular depot injection at in two different dose levels (dose level 1 and dose level 2) and collected the blood at different time points to measure the plasma concentration of Atovaquone and Proguanil. The plasma exposure of Atovaquone and Proguanil measured at various time points and presented in table 1 and table 2.
Table-1: Estimation of concentration of Atovaquone and Proguanil in plasma samples of three rats from treatment group 1 (Dose level 1) after administration of 15.57mg/kg of Atovaquone and 10.83 mg/kg of Proguanil.

Time [h] Atovaquone [µg/ml] Mean[µg/ml] SD
#1 #2 #3
24 9.91 8.75 6.17 8.28 1.92
168 5.99 5.88 11.99 7.95 3.50
240 10.42 10.78 7.53 9.58 1.78
312 10.44 10.74 6.17 9.12 2.55
Time [h] Proguanil [ng/ml] Mean [ng/ml] SD
24 100 103 45.6 82.94 32.38
168 26.0 25.8 13.9 21.91 6.94
240 9.69 11.2 BLQ 10.42 NA
312 9.26 11.5 BLQ 10.34 NA
BLQ : Below the limit of quantification
SD: Standard deviation
NA: Not applicable.
Table 2: Estimation of concentration of Atovaquone and Proguanil, in plasma samples of three rats from treatment group 2 (Dose level 2) after administration of 7.8mg/kg of Atovaquone and 5.4 mg/kg of Proguanil.
Time [h] Atovaquone [µg/ml] Mean[µg/ml] SD
#1 #2 #3
24 3.96 1.46 1.84 2.42 1.35
168 9.80 2.45 2.48 4.91 4.23
240 3.82 13.33 12.35 9.83 5.23
312 3.65 13.54 12.72 9.97 5.49
Time [h] Proguanil [ng/ml] Mean [ng/ml] SD
24 61.2 41.7 35.7 46.17 13.32
168 BLQ 4.10 4.85 4.48 4.10
240 BLQ BLQ BLQ NA NA
312 BLQ BLQ BLQ NA NA

As demonstrated above, the novel depot injectable formulation of Atovaquone and Proguanil provided according to the present invention sustains the therapeutic effect of Atovaquone and Proguanil for at least 2 weeks, preferably 4 to 8 weeks and more preferably 12 weeks thereby offering prolonged therapeutic and prophylactic benefit. Therefore, novel depot injectable formulation of Atovaquone and Proguanil provided according to the present invention is useful as a prophylactic treatment for travelers to malaria-endemic countries.

Example 5:
Spray Dried Atovaquone Formulation Composition -8
Taken Spray Dried Atovaquone powder 4.76 gms (which contains 3.3 gms Atovaquone) and dissolved in 20 ml of N-Methyl-2-pyrrolidone. In another taken 38 ml castor oil and dissolved in N-Methyl-2-pyrrolidone(30ml). Added the Spray Dried Atovaquone solution into the castor oil solution, adjusted the quantity sufficient to make up the volume upto 100 ml with NMP and stirred well for an hour to obtain clear solution.

Atoguanil Spray Dried Formulation Composition
Ingredients Quantity
Atovaquone 3.3 gm
Proguanil NIL
Castor Oil 38 ml
Kolliphor HS 15 1.323 gm
PVP-K30 0.047 gm
poloxamer 188 0.095 gm
NMP Q.S to 100 ml